MONITORING OF INFLUENZA A AND B VIRUSES IN ITALIAN WASTEWATER DURING THE 2022-2023 SEASON: A COMPARATIVE ANALYSIS WITH CLINICAL SURVEILLANCE DATA

Aim of the Study: Influenza, an acute respiratory infection caused by the influenza viruses, is a highly infectious respiratory disease with a significant global impact, resulting in millions of cases and hundreds of thousands of deaths annually, according to the World Health Organization. The main types, influenza A virus (IAV) and influenza B virus (IBV), cause seasonal outbreaks. Several studies have shown that these viruses can be detected in wastewater. Therefore, wastewater-based epidemiology (WBE) provides a valuable tool for monitoring pathogen transmission in communities. The aim of this study was to detect and quantify IAV and IBV in wastewater from October 2022 to April 2023. Environmental data were compared with clinical surveillance data from RespiVirNet, an integrated surveillance system that tracks respiratory viruses in Italy. Methods: We first optimised real-time PCR primers/probes developed by the Centers for Disease Control and Prevention (CDC) adapting them for use with digital PCR, following the Digital MIQE Guidelines. We then proceeded with testing of environmental samples. A total of 298 wastewater samples were collected from 67 wastewater treatment plants (WTPs) in 20 Italian regions and autonomous provinces during the influenza season, from October 2022 to April 2023. Quality assurance controls (including process control virus and inhibition control) were incorporated to evaluate viral recovery and PCR inhibition. The samples were tested for both influenza A and B using a multiplex assay. Results: Of the 298 analysed samples, 19% tested positive for Influenza A, and 17% tested positive for Influenza B. Influenza A gradually increased from October to December, peaking at 61% in December, while Influenza B showed a slight increase in November (15%) and peaked at 36% in February 2023. For Influenza A, concentrations ranged from 3,9E+05 to 7,7E+07 genome copies (g.c.)/L of wastewater, while for Influenza B concentrations varied between 3,9E+05 to 5,7E+06 g.c./L of wastewater. The peak of positive cases for Influenza A (46% of positive samples) corresponded to the peak of environmental data (week 48 of 2022), after which it steadily and consistently declined until the end of surveillance (week 14 of 2023). On the other hand, the percentages of positive cases for Influenza B remained very low until February 2023, with a peak reached around week 9 of 2023 (14,4%), aligning with the environmental data. Conclusions: In general, the environmental results were consistent with the clinical surveillance trends observed during the study period, thereby demonstrating the value of WBE in tracking epidemiological patterns and informing public health interventions. This is particularly relevant in light of the proposed revision of the Urban Wastewater Treatment Directive 91/271/EEC, which emphasizes the need for Member States to monitor public health parameters in urban wastewater, including influenza viruses. This reflects the increasing recognition of the importance of wastewater in influenza surveillance and wider public health monitoring. Acknowledgements: This research was supported by EU funding within the NextGeneration EU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, PE13 INF-ACT) and by the CCM project ‘Respiratory Virus Surveillance System in Wastewater’ funded by the Ministry of Health